1. Field of the Invention
Parkinson's disease (PD) is a common neurodegenerative disorder with a lifetime incidence of approximately 2 percent. A pattern of familial aggregation has been documented for the disorder, and it was recently reported that a PD susceptibility gene in a large Italian kindred is located on the long arm of human chromosome 4. We have identified a mutation in the alpha synuclein gene, which codes for a presynaptic protein thought to be involved in neuronal plasticity, in the Italian kindred and in three unrelated families of Greek origin with autosomal dominant inheritance for the PD phenotype. This finding of a specific molecular alteration which is causative for PD will permit the detailed understanding of the pathophysiology of the disorder. In addition, methods of screening nucleic acids for the presence of mutations in the synuclein gene to test for predisposition to Parkinson's Disease are now possible.
2. Technology Background
Parkinson's disease (PD) was first described by James Parkinson in 1817 (1). The clinical manifestations of this neurodegenerative disorder include resting tremor, muscular rigidity, bradykinesia and postural instability. A relatively specific pathological feature accompanying the neuronal degeneration is the intracytoplasmic inclusion body, known as the Lewy body, which is found in many regions including the substantia nigra, locus ceruleus, nucleus basalis, hypothalamus, cerebral cortex, cranial nerve motor nuclei, and the central and peripheral divisions of the autonomic nervous system (1).
In many cases a heritable factor predisposes to the development of the clinical syndrome (2). We have recently shown that genetic markers on human chromosome 4q21–q23 segregate with the PD phenotype in a large family of Italian descent (3). The clinical picture of the PD phenotype in the Italian kindred has been well documented to be typical for PD, including Lewy bodies, with the exception of a relatively earlier age of onset of illness at 46±13 years. In this family the penetrance of the gene has been estimated to be 85%, suggesting that a single gene defect is sufficient to determine the PD phenotype.
We now report the identification of a mutation in the alpha synuclein gene that is associated with Parkinson's disease. The mutation, an Ala53Thr substitution, was found to be linked to the PD phenotype in four independent PD families and absent from 314 control chromosomes, providing strong genetic evidence that this mutation in the human alpha synuclein gene is causative for the PD phenotype in these families.
The Ala53Thr substitution is localized in a region of the protein whose secondary structure predicts an alpha helical formation, bounded by beta sheets. Substitution of the alanine with threonine is predicted to disrupt the alpha helix and extend the beta sheet structure. Beta pleated sheets are thought to be involved in the self aggregation of proteins which could lead to the formation of amyloid like structures (6).
This was already tested in the case of NAC35, the 35 amino acid peptide derived from alpha-synuclein that was first isolated from plaques found in patients with Alzheimer's disease (4). NAC35 was shown to self aggregate and form amyloid fibril which shared the ‘amyloid’ characteristics of insolubility in aqueous solutions and green birefringence under polarized light, subsequent to Congo red staining (6). NAC35 is located in the middle of the alpha synuclein molecule and extends from amino acid 61 to amino acid 95. Residue 53, which is found to be mutated in PD, is outside the NAC35 peptide found in amyloid plaques. However, the true size of the NAC peptide involved in the plaques is not known since the protease used to isolate the peptide from AD tissue cuts at lysine 60 of the alpha synuclein protein. It is therefore possible that amino acid 53 may be part of the NAC peptide found in plaques. In crosslinking experiments with beta amyloid (Abeta), it was demonstrated (6) that residues 1–56 and 57–97 specifically bind amyloid and that a synthetic peptide consisting of residues 32–57 performed similarly.
Three members of the synuclein family have been characterized in the rat, with SYN1 exhibiting 95% homology with the human alpha-synuclein protein (7). SYN 1 of the rat is expressed in many regions of the brain, with high levels found in the olfactory bulb and tract, the hippocampus, dentate gyrus, habenula, amygdala and piriform cortex, and with intermediate levels in the granular layer of the cerebellum, substantia nigra, caudate-putamen, and dorsal raphe (7). This pattern of expression coincides with the distribution of the Lewy bodies found in brains of patients with Parkinson's disease. It is also interesting to note that decrease in olfactory sense often accompanies the syndromic features of Parkinson's disease, and in many cases it is proposed that hyposmia is a prodromic sign of the illness (8).
In the zebra-finch the homologue to alpha synuclein, synelfin, is thought to be involved in the process of song learning, suggesting a role for synuclein perhaps in memory and learning (9). In contrast to humans, rats have a threonine at residue 53 of their homologues to the human alpha synuclein gene (FIG. 4). Similarly, the zebra-finch synelfin carries a threonine at amino acid 53, whereas both Bos taurus and Torpedo californica do not (10). There are no reports that suggest the presence of Lewy bodies in the brains of the rat or the zebra finch or a phenotype resembling that of PD. Lack of any phenotype could be explained by a combination of factors, including the following: the relative short life span of rodents may prohibit the observation of a late onset disorder, interaction with other cellular components not present in the rat may be required for the phenotype, absence of a critical environmental trigger in the rodents, or finally a heterozygous status Ala/Thr may be necessary for the production of a phenotype.
Studies of early onset AD have previously documented that missense mutations can cause an adult onset neurodegenerative disorder. Of the 31 mutations described so far in the loci for presenilin 1 and 2, thirty were missense and one was a splice variant (11). Missense mutations in the prion protein have also been implicated in the amyloid production seen in Gerstmann-Straussler-Scheinker and Creutzfeld-Jakob diseases, both forms of spongiform encephalopathy (12). Studies in these neurodegenerative disorders have pointed to the importance of the physical chemical properties of mutant cellular proteins in initiating and propagating neuronal lesions leading to disease. Similar studies in the synuclein protein family may provide valuable insights into the etiology and pathogenesis of PD.
Similarly with the mutations in the presenilin genes in patients with early onset Alzheimer's disease, the mutation identified in the alpha synuclein gene is unlikely to account for the majority of sporadic and familial cases of PD. However, this mutation may account for a significant proportion of those families with a highly penetrant, early onset autosomal dominant PD phenotype.
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